1337-C - Live Cell 3D HCS Assay Development and Validation Using a Tumor Organoid Model of EMT in Colon Cancer

Tuesday, February 6, 2018

2:00 PM - 3:00 PM

Epithelial-mesenchymal transition (EMT) is linked to the pathology of the most prominent human diseases such as fibrosis and cancer. Overwhelming evidence from the collective literature has been building for decades implicating EMT as a driving force for tumor progression and metastasis. Although our biological knowledge foundation of EMT is firm, the cellular changes that govern EMT are complex, involving cues from the microenvironment, multiple signaling pathways, and a multitude of gene expression changes. Constructing a more complete chemical biology knowledge base of EMT is of significant importance that will lead to a better understanding of EMT driven disease and help to develop more effective drug therapies. Here, we describe the development and validation of a live-cell 3D multicellular tumor spheroid (MCTS) model for high content screening (HCS) EMT probe discovery program. This phenotypic-based assay design features a lentiviral vector backbone with dual reporter of E-cadherin promoter (EcadPro–mCherry fluorescent protein) and vimentin promoter (VimPro–EGFP fluorescent protein) expressed in self-assembled organoids using SW620 colorectal cell line. The outcome of this dual reporter expression is engineered to identify compounds that inhibit, modulate, or reverse EMT in real-time. The supplementary addition of two bioprobes, Hoechst® 33342 and DRAQ7™ provides detection of cell nuclei for segmentation and cell viability respectively. We have initially developed and validated this model in a primary HCS screen against a focused pilot compound library of 2,550 compounds using the Operetta CLS™ high content imager by capturing multiple 3D z-stack image planes and subsequent image analysis of the 3D image stack.

Primary Author - February Poster(s)

Joe Trask

Senior Application ScientistPerkinElmer, Inc.Bahama, NC

Joe is instrumental in strategic customer support through teaching, training, and collaborations at PerkinElmer Cellular Imaging and Analysis group. He brings over 20 years of experience in high content screening technology from academia (Duke University and The Ohio State University), pharmaceutical industry (Abbott Laboratories and Sphinx Laboratories, Division of Eli Lilly & Company), and biotechnology (The Hamner Institute for Health Sciences and ScitoVation LLC). Joe has extensive experience in cell based technologies from flow cytometry, confocal microscopy and computer-assisted automated microscopy studying cancer, immunology, neurodegeneration, and toxicology. Joe has published several research articles and book chapters in High Content Imaging field, and is an author and co-editor to the NIH/NCATS Assay Guidance Manual. Joe was a co-founder and first President of Society for Biomolecular Imaging and Informatics (SBI2) and is a co-editor of a recent book “High Content Screening: A Powerful Approach to Systems Cell Biology and Drug Discovery”.